The aim of this project is to identify and characterize factors that regulate gene expression in vertebrate embryos as an approach to understanding the mechanisms that control early development. The primary model system is an epidermal keratin gene, XK8lAl, expressed in the amphibian (Xenopus laevis) embryo. The upstream cis-regulatory region of this gene has been mapped by introducing mutated DNA constructs into frog embryos. Within this regulatory domain, we have identified a high-affinity binding site for the Xenopus homolog of a mammalian transcription factor, AP-2 (XAP-2). We have cloned XAP-2, and shown that binding of this factor to the XK8lAl regulatory region is necessary for efficient expression of microinjected DNA. We have also demonstrated that XAP-2 RNA and protein are located in both epidermal and neural crest cells in early development. The XAP-2 target gene, keratin XK81AI, is only active in epidermis; this and other data suggest that additional factors must cooperate with and modulate the function of XAP-2 in the early embryo. We are attempting to identify these factors. In addition to XK8lAl, we are studying the regulation of two other ectoderm-specific genes; XAP-2 and Xenopus Distal-less 2 (XD11-2). The latter, a Xenopus homolog of a Drosophila homeobox gene, was cloned by M. Jamrich's laboratory at FDA, with whom we are collaborating. XD11-2 is expressed specifically in embryonic and adult epidermis, in both frog and mouse. We have isolated Xenopus genomic clones for both genes, and we are in the process of carrying out regulatory mapping experiments similar to those performed with XK8lAl.